Photochemistry and Photobiology, 2005, 81 : 183-1 86

Research Note

The Separation of Hypericin’s Enantiomers and Their Photophysics in Chiral Environments?

Lindsay Sanders, Mintu Halder, Tom Ling Xiao, Jie Ding, Daniel W. Armstrong and Jacob W. Petrich* Department of Chemistry, Iowa State University, Ames, IA

Received 28 May 2004; accepted 8 July 2004

ABSTRACT

We report the first separation of the enantiomers of hypericin. Their steady-state optical spectra and ultrafast primary photoprocesses are investigated in chiral environments. Within experimental error, there is no difference between the two enantiomers in any of the systems considered. This is consistent with the emerging picture that the rich and extended absorption spectrum of hypericin is not a result of ground-state heterogeneity. It is also consistent with the observation that the spectra and photophysics of hypericin are generally insensitive to environments in which it does not aggregate.

I NTRO DU CTlO N This article focuses on the fluorescence spectroscopy of the two enantiomers of hypericin in chiral environments. Hypericin (Fig. 1) is a naturally occumng perylene quinone that has gained great interest recently because of its biological activity (1-20), in particular, its light-induced biological activity (21-25). The importance of light for its function has motivated our study of the photophysics of hypericin and its analogs (26-36). Two notable features of the steady-state spectra of hypericin are the mirror image symmetry between the absorption and emission spectra and the extended absorption from the visible to the ultraviolet with no gaps of zero absorbance. Our first attempt to explain these features was to suggest that the ground state of hypericin was populated with at least one other species, for example, a monotautomer (27). However, temperature-dependent ‘H nuclear magnetic resonance (NMR) and two-dimensional rotating-frame Overhauser enhance- ment spectroscopy studies of hypericin show that there is only one conformer or tautomer for hypericin in the ground state (37). Ab

YPosted on the website on 13 July 2004. *To whom correspondence should be addressed: Department of

Chemistry, Iowa State University, Ames, IA 5001 1-3111, USA. E-mail: jwp@iastate.edu

Abbreviations: DMSO, dimethylsulfoxide; GST, glutathione S-transferase; HSA, human serum albumin; NMR, nuclear magnetic resonance; PMT, photomultiplier tube; TEAA, triethylamine.

0 2005 American Society for Photobiology 003 1-8655/05

initio quantum mechanical calculations are consistent with this observation (38). In the spirit of continuing to search for instances of ground-state heterogeneity, we have separated hypericin’s enantiomers (Fig. 1): the methyl and hydroxyl groups in the so- called bay region impose right- and left-handed twists about the axis containing the carbonyl groups. We have investigated their steady-state spectra and ultrafast primary processes.

MATERIALS AND METHODS Separation of hypericin enantiomers. Separations and collections of enantiomers of hypericin were achieved using a HP 1050 high-performance liquid chromatography (Agilent Tech, Palo Alto, CA) system with UV detector, auto injector and a computer-controlled Chem-station data processing software. The chiral stationary phase, trade named Chirobiotic TAG column (250 X 4.6 mm inner diameter) was obtained from Advanced Separation Technologies Inc. (Whippany, NJ). The chiral stationary phase was prepared by bonding the chiral selector to a 5 pm spherical porous silica gel through linkage chains. Detection wavelengths were varied between 220 and 254 nm to confirm the same absorption ratio of the two enantiomers. The semipreparative separation conditions used to isolate microgram quantities (overall) of the pure enantiomers of hypericin are as follows. The racemates of hypericin were dissolved in neat methanol to a concentration of 1 mg/mL. Up to 50 pL of sample was injected onto an analytical scale Chirobiotic TAG column and eluted with 100% methanol, adding 1% triethylamine (TEAA)(vol/vol). Fractions of the individual enantiomers (from successive injections) were collected manually and concentrated by evaporation at room temperature using continuously flowing air (21°C). All mobile phases were premixed and degassed before use. The flow rate was 0.4 mL/min under isocratic conditions. We did not determine the absolute configurations of the separated enantiomers, which are referred to as hypericin 1 (13.83 min elution time) and 2 (16.65 min elution time) (Fig. 2) .

Sample preparation for optical measurements. A tightly closed 3 mm pathlength quartz cuvette (instead of a traditional 10 mm cell) was used for all spectroscopic measurements because of the limited amount of sample available to us. All measurements were taken at ambient temperature. Steady-state excitation and emission spectra were recorded with a SPEX Fluoromax (SPEX Industries Inc., Edison, NJ) with a 4 nm handpass and were corrected for detector response. The excitation wavelength for the emission spectra presented in Fig. 3 was 550 nm. The emission monochrometer was set at 650nm for the excitation spectra. Solutions of each hypericin enantiomer (5.0 X lo-’ M each) were prepared in (S)-(+)-2- butanol(99%) purchased from Aldrich (Milwaukee, WI). A Concentrated y- cyclodextrin (99%, Sigma, St. Louis, MO) solution was prepared in water. The chiral hypericins were dissolved in aliquots of this solution and diluted to 1.0 X lo-’ M . Human serum albumin (HSA, 99%) was purchased from Sigma. Solutions of 1.0 X M hypericin with 5.0 X 10” M HSA were prepared in a 7 mM phosphate buffer (pH 7) using concentrated stock solutions of hypericin in dimethylsulfoxide (DMSO). Microliter quantities of

183

184 Lindsay Sanders eta/,

Figure 1. Structures of hypericin enantiomers.

hypericin in DMSO were used to keep the final DMSO concentration below 0.8%. The solutions were allowed to equilibrate for 24 h at 4'C in the dark. Solutions of 5.0 X M hypericin with 2.5 X M glutathione S- transferases (GST) were prepared in 7 mM phosphate buffer (pH 7) with 2 mM ethylenediaminetetraacetic acid. Concentrated stock sotutions of hypericin in DMSO were added in microliter quantities so that the final DMSO concentration remained below 3%. The solutions were left to equilibrate for 24 h at 4°C in the dark.

Fluorescence upconversion measurements. The fundamental output from a homemade Ti-sapphire amplified system (39,40) (815 nm) is doubled by a Type-I lithium triborate crystal (2 mm). The frequency-doubled blue pulses (407 nm) are separated from the fundamental by a dielectric mirror coated for 400 nm and are focused onto a rotating cell containing the sample using a 5 cm convex lens. The remaining fundamental was used as the gate to upconvert emission. Fluorescence was collected by an LMH- 1OX microscopic objective (OFR Precision Optical Products, Caldwell, NJ) coated for near UV transmission. The gate and the emission are focused by a quartz lens (12 cm) onto a Type-I 0.4 mm P-barium borate crystal (MgF2 coated, cut at 3 1" and mounted by Quantum Technology Inc., Lake Mary, FL). The polarization of both the gate and excitation source was controlled with a set of zero-order half-wave plates for 800 and 400 nm, respectively. The upconverted signal is then directed into an H10 (8 nm/mm) monochromator (Jobin Yvon/Spex Instruments S. A. Group, Edison, NJ) with a 5 cm convex lens coupled to a Hamamatsu R 980 photomultiplier tube (PMT) equipped with a UGll UV-pass filter and operated at maximum sensitivity. The PMT output was amplified in two stages by a Stanford research Systems (Sunnyvale, CA) SR-445 DC-300 MHz amplifier with input terminated at 500 and was carefully calibrated after a long (1-2 h) warm-up. Photon arrival events were registered with SR-400 gated photon counter operated in continuous wave mode with a threshold level of -100 mV. This signal was fed into boxcar averager. A part of blue pulse train was used to normalize pump beam fluctuations. A translation stage with a resolution of 0.06 mm/step was used to delay the exciting pulses and a computer with Keithley Metrabyte (Cleveland, OH) (DAS 800) interfacing card for driving the motor. The instrument response function was obtained by collecting the cross-correlation function of the blue and red pulses; the resulting third harmonic intensity was plotted against delay time. The cross-correlation functions typically have a full width at half maximum of - 1 ps. This instrument response is a little over three times as broad as that obtained with our unamplified system (34,41). We attribute this to the absence of compensating prisms after frequency

Abs 13.83 16.65

~ Time (minutes)

Figure 2. Chromatogram obtained using 100% MeOH, 1% TEAA (vov vol), 0.4 mL/min on Chirobiotic TAG column for separation of racemic hypericin.

0

350 450 550 650 750

Wavelength (nm) Figure 3. Steady-state excitation and emission spectra of hypericin 1 (solid line) and hypericin 2 (dashed line) in (a) (S)-2-butanol; (b) y-cyclodextrin, (c) HSA and (d) GST. For all excitation spectra, the emission was collected at 650 nm; and for all emission spectra, the excitation monochromator was set at 550 nm.

doubling, the presence of the rotating sample cell and perhaps a nonideal optical geometry, which nevertheless permits the facile interchange between pumpprobe transient absorption and fluorescence upconversion measurements. All curves were fit and deconvoluted from the instrument function using an iterative convolute-and-compare least squares algorithm.

RESULTS AND DISCUSSION The steady-state spectra (Fig. 3) of the hypericin enantiomers were obtained in the following chiral environments: (S)-(+)-2-butanol, y- cyclodextrin, HSA and GST. The latter two are of particular biological interest. HSA is a transport protein in the blood plasma. It binds a wide variety of substances, such as metals, fatty acids, amino acid, hormones, and a large number of therapeutic drugs (42). Be- cause of its clinical and pharmaceutical importance, the interaction of HSA with ligands has been studied (43,44); and, in particular, the interaction of hypericin with HSA has been investigated (4549). GST are a family of detoxification enzymes that are known to bind nonsubstrate hydrophobic anions such as hemes and porphyrins (50). It has been demonstrated recently that two forms of GST bind hypericin very tightly: the form studied here, Al-1, binds hypericin with KD = 70 nM. Although there are slight differences in the hypericin spectra with respect to the four different environments, within experimental error, the two hypericin enantiomers yield identical spectra when they are in the same chiral environment.

Figure 4 presents the fluorescence upconversion traces for hypericin in S-2-butanol on a 100 ps timescale. The rising component is the signature we have attributed to excited-state intramolecular H-atom transfer (5 1). Within experimental error, this component is present for racemic hypericin in DMSO and for the hypericin enantiomers in S-2-butanol in the same amount and with the same time constant. This is in stark contrast to the recent study where it has been shown that 5,8-dicyano-2-naphthoI has different emission spectra and excited-state proton transfer kinetics in either R- or S-2-butanol compared with that in racemic 2-butanol (52).

CONCLUSIONS Falk and coworkers have separated diastereomeric derivatives of hypericin and have discussed the barrier to their interconversion

Photochemistry and Photobiology, 2005, 81 185

1.2=

1.0 -

0.8 -

0.6 -

0.4 -

3 v U

Hypericin-2 in (s)-2-Butanol

Hypericin-l in (s)-2-Butanol I ‘Hypericin in DMSO 1

0.2 +q 600 nm 0.0

I I I

0 20 40 60 80

Time (ps) Figure 4. Fluorescence upconversion trace for hypericin in DMSO and for the two hypericin enantiomers in S-2-butanol at 600 nm. he, = 407 nm. The traces are offset vertically with respect to each other to facilitate visual comparison. They can be fit globally with a rising component of 11 ps. This component represents about 30% of the signal.

(53-55). We present in this study the first direct separation of the enantiomers depicted in Fig. 1. This study was motivated by the question of whether the rich and complicated spectra of hypericin can be attributed to ground-state heterogeneity. Ab initio quantum mechanical calculations (38) and NMR experiments (37,56) suggest that there is only one species in the ground state. Within experi- mental error, there is no difference between steady-state spectra of the two enantiomers in any of the systems considered: (9-(+)-2- butanol, y-cyclodextrin, HSA and GST. Nor is there any difference in the excited-state H-atom transfer kinetics of the two enantiomers in (S)-(+)-2-butanol. This is consistent with the emerging picture that the rich and extended absorption spectrum of hypericin is not a result of ground-state heterogeneity. It is also consistent with the observation that the spectra and photophysics of hypericin are generally insensitive to environments in which it does not aggregate.

Acknowledgements-D.W.A was supported by the National Institutes of Health, N M R01 GM53825-06. We thank Mr. Pramit Chowdhury for technical assistance. We thank Prof. W. Atkims and Mr. Doug Lu for sam- ples of the GST and for furnishing its binding constant with hypericin.

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